Short-term physiologic response of the green microalga Picochlorum sp. (BPE23) to supra-optimal temperature

Photobioreactors heat up significantly during the day due to irradiation by sunlight. High temperatures affect cell physiology negatively, causing reduced growth and productivity. To elucidate the microalgal response to stressful supra-optimal temperature, we studied the physiology of Picochlorum sp. (BPE23) after increasing the growth temperature from 30 °C to 42 °C, whereas 38 °C is its optimal growth temperature. Cell growth, cell composition and mRNA expression patterns were regularly analyzed for 120 h after increasing the temperature. The supra-optimal temperature caused cell cycle arrest for 8 h, with concomitant changes in metabolic activity. Accumulation of fatty acids was observed during this period to store unspent energy which was otherwise used for growth. In addition, the microalgae changed their pigment and fatty acid composition. For example, palmitic acid (C16:0) content in the polar fatty acid fraction increased by 30%, hypothetically to reduce membrane fluidity to counteract the effect of increased temperature. After the relief of cell cycle arrest, the metabolic activity of Picochlorum sp. (BPE23) reduced significantly over time. A strong response in gene expression was observed directly after the increase in temperature, which was dampened in the remainder of the experiment. mRNA expression levels associated with pathways associated with genes acting in photosynthesis, carbon fixation, ribosome, citrate cycle, and biosynthesis of metabolites and amino acids were downregulated, whereas the proteasome, autophagy and endocytosis were upregulated.

Photobioreactors heat up significantly as a result of irradiation by sunlight, leading to shifts in temperature throughout the day up to levels that can be stressful for microalgae. Heat stress poses a significant challenge in the cultivation of microalgae for the production of food, feed and chemicals. Non-optimal temperatures reduce microalgal growth, thereby reducing productivity and photosynthetic efficiency 1 . The effect of excessive heat on the cellular physiology has been studied in photosynthetic model organisms such as Arabidopsis thaliana and Chlamydomonas reinhardtii. While many aspects of the response to heat have been studied, much still has to be learned on how exactly microalgae cope with heat [2][3][4] . In addition, detailed research on the effect of heat stress on other photosynthetic (micro)organisms, is scarce.
Many cellular processes are impacted by supra-optimal temperature which ultimately unbalances cellular homeostasis 5 . Several effects are; failure of correct protein folding and protein complex assembly, destabilization of cell membranes through increased membrane fluidity, a reduction in photosynthesis, changes in DNA replication and repair, changes in enzymatic activity, and reduced metabolic activity 2,6,7 . As a response to temperatureinduced physiological changes, heat shock factors (HSF's) and heat shock proteins (HSP's) are expressed to mediate and protect against adverse effects through chaperoning after exposure to heat stress 4,5,8 . Excessive heat kills microalgae quickly due to denaturation of proteins and destabilization of membranes. However, in this paper we will focus on a supra-optimal temperature. Supra optimal temperatures fall between the optimal and maximal temperature for growth. Here, growth rate is reduced, but cells still survive.

Results
Temperature increase to supra-optimal level leads to cell-cycle arrest and decreased growth. The effect of the increase in temperature from 30 to 42 °C for a time span of 120 h on Picochlorum sp. (BPE23) can be seen in Fig. 1. The specific growth rate, quantum yield, and cell volume were measured over time, before and after temperature increase. At the onset of temperature increase (t = 0), the cell culture was at steady state, in a photobioreactor operated in turbidostat mode. Since only temperature was changed, it is safely assumed that the observed changes in the culture are solely due to the temperature shift. Two distinct responses were observed; an immediate short-term response, followed by a long-term response in which the microalgae gradually acclimated. www.nature.com/scientificreports/ One hour after the temperature shift all monitored growth parameters had changed (Fig. 1). The specific growth rate decreased during the first hour, followed by a temporary increase to higher levels than those observed at 30 °C. It remained high until 8 h after the temperature shift point at which it started to decrease until the end of the experiment (120 h). During the first 8 h the average cell volume increased by 30%, from 10.6 to 13.76 µm 3 . The combination of a decreased growth rate and an increase in cell volume due to halted cell division indicates a cell cycle arrest. The cell cycle arrest was released after 8 h and cell volume returned to the initial value after 24 h. The cell volume gradually kept increasing from 24 h after the temperature shift until the end of the experiment.
Quantum yield (F v /F m ) is a measure for the maximum photosynthetic capacity of photosystem II and can be used to monitor photo inhibitory damage in response to stress, in this study as a result of high temperature 18 . The observed quantum yield shortly increased from 0.72 to 0.75 at 42 °C, after which a gradual decrease was observed to a level of 0.45 at 72 h and remained stable until the end of the experiment (120 h).
Pigment content increased after exposure to supra-optimal temperature. Pigment content was measured over time and is displayed in Fig. 2. The pigment concentrations were also measured through optical density measurements which show a comparable trend (Supplementary material 5). The concentration of chlorophyll-a and chlorophyll-b decreased slightly in the first hour after the temperature shift. However, from 4 h onwards an increase in concentration was observed with a peak at 72 h. The concentration for both chlorophyll-a and chlorophyll-b increased with a factor of 1.75, indicating an upregulation of photosystems. The ratio between chlorophyll-a and chlorophyll-b increased slightly between 8 and 24 h, but returned to the values as found at the start of the experiment at 72 h.
Carotenoids, in addition to a light-harvesting function within the photosystems, also have a photoprotective role in non-photochemical quenching, ROS scavenging, and/or filtering of light 19 . The xanthophyll pigments Violaxanthin and Zeaxanthin show a peak concentration at 72 h after temperature increase. Antheraxanthin was measured but not detected. The concentration of zeaxanthin increased directly after exposure to supra-optimal temperature, from 0.18 to 1.84 mg g −1 after 72 h, whereas the concentration of violaxanthin initially decreases after 1 h, followed by a gradual increase during the first 24 h. Xanthophyll pigments accommodate energy dissipation through non-photochemical quenching 19 . Also, β-carotene, Canthaxanthin and Lutein, known as strong antioxidants, were found at increased concentrations in stressed cells after exposure to supra-optimal temperatures 19 .
Picochlorum sp. (BPE23) accumulated fatty acids as a response to the increased temperature. Fatty acids can be distributed into two general groups; neutral and polar, which are present as lipid bodies and in the cell membranes, respectively (Fig. 3). The concentration of neutral fatty acids increased from 3.0 mg g −1 at 0 h to 11.5 mg g −1 at 24 h, whereas the total content of polar fatty acids increased from 79.0 to 88 mg g −1 . Within the neutral fatty acids, all fatty acid species showed an increased concentration at approximately the same ratio. In the polar fatty acids the increase in content was mainly caused by C16:0 content, which C16:0 increased from 21.0 mg g −1 after 0 h to 30.0 mg g −1 after 72 h. At the same time, a decrease in C16:3 was observed over time.
A large transcriptional change was observed after the increase in temperature. Comparative transcriptome analysis provided further insight into the stress response of Picochlorum sp. (BPE23) to supraoptimal temperatures. In total 6990 genes were identified in the transcriptome, of which 5930 were annotated through BLASTP and Orthofinder search and inferring domains. In addition, 55 novel genes were identified. 4427 of these genes matched to A. thaliana proteins (62.8%). Iteratively, the remaining unidentified genes were www.nature.com/scientificreports/ matched to other species of which 678 genes matched to C. Variabilis orthologs (9.6%), 116 genes matched to A. protothecoides orthologs (1.6%), 3 genes matched to Helicosporidium sp. orthologs (0.04%) and 60 genes matched to C. reinhardtii orthologs (0.9%). 643 out of the remaining 1761 genes had a domain annotation (9.1%). Differential gene expression levels and enrichment analyses are displayed in two ways in Figs. 4C,D, 5, and 6. The left-hand side of Fig. 4 shows the number of genes that are differentially expressed in the individual timepoints compared to the control condition at timepoint zero, which represents steady state growth at 30 °C. The right-hand side of the figure shows the number of genes that are differentially expressed between consecutive timepoints.
39% of all genes showed an log2 fold change value larger than 1 after 1 h, which was largely reversed between 1 and 4 h (Fig. 4). The number of differentially expressed genes (DEGs) decreased from 4 to 8 h, and subsequently to 24 h after the temperature increase. This indicates that the initial heat stress subsided and that Picochlorum sp. (BPE23) was reverting to a stable state once again. Between 24 and 120 h only 2% of genes were differentially expressed. The changes in log2 fold change after 8 h, 24 h and 120 h look very similar when compared to 0 h. However, when compared to the previous sampling time then differences in expression levels can be observed.
A PCA was conducted to identify similarities among biological replicates (Fig. 4). mRNA expression at 0 h and 1 h showed a significant distance from samples taken at 4  Temperature stress affects protein processing, HSP expression, and DNA replication. KEGG pathway enrichment was performed to identify which metabolic pathways were affected by the temperature increase (Fig. 5). The RNA degradation pathway was upregulated directly after temperature increase. Other upregulated pathways involved in the immediate response were the proteasome, autophagy, and endocytosis pathways. Downregulated pathways were DNA replication, ribosome, and base/nucleotide excision repair. In addition, pathways in the central metabolism such as biosynthesis of amino acids, biosynthesis of secondary metabolites, and citrate cycle were downregulated 1 h after the temperature increase. A reversal of this initial downregulation was observed after 4 h. Despite the observed upregulation between sampling moments, the pro- www.nature.com/scientificreports/ cesses were never enriched in the upregulated pathways when compared to growth at 0 h (30 °C). The ribosome pathway remained enriched in the downregulated pathways compared to timepoint zero throughout the experiment. Photosynthesis pathway was downregulated after 8 h. Despite enrichment in the upregulated pathways at 24-8 h, the photosynthesis was still downregulated compared to 0 h. From 24 to 120 h after the temperature shift, downregulation increased further. GO-term enrichment analysis was conducted in addition to the KEGG enrichment to further identify enriched cellular processes (Supplementary material 2). Various GO-terms related to protein processing such as Protein folding, Proteolysis and Proteasome were upregulated throughout the experiment. These GO-terms showed enrichment in the upregulated GO-terms compared to the control at 0 h. In addition, cellular response to heat and heat shock protein binding show enrichment in the upregulated GO-terms. On the contrary, DNA replication, photosynthesis, peptide biosynthesis, translation, and three ribosome related GO-terms are enriched in the downregulated genes. The observations from the GO-term enrichment indicate similar changes in the mRNA expression as the observations from the KEGG pathway enrichment indicated. . Each module was functionally annotated with GO-terms and KEGG pathways to assess differences between modules (Supplementary material 4). Each module displayed a unique functional annotation pattern which consisted of multiple GO-terms. The hubs were grouped roughly by similarities in functionality. The different module colours and their phylogenetic relationships are displayed in Fig. 6. The brown, cyan, green, grey and yellow modules were made up of genes associated with proteolysis, protein ubiquitination, autophagy, endocytosis and protein processing in ER. These processes are involved in the protein-related protection response to temperature stress 4 . Furthermore, the light-green, black, green-yellow and turquoise modules were associated with processes to energy fixation and metabolism, such as ATP binding, oxidative phosphorylation, photosynthesis, thylakoid membrane, carbon fixation, glycolysis and gluconeogenesis. Other interesting modules which were affected by temperature stress are the blue, light-cyan, magenta, pink and red module. These modules are associated with RNA-, and DNA-related processes such as replication, binding and repair. Lastly, all hubs except for grey and yellow were associated to the chloroplast envelope, chloroplast stroma, and plastid. The average module expression profile was summarised through the module's eigengene. The eigengene is a hypothetical gene that is highly correlated with expression profiles of genes in the module and can therefore function to represent the average expression of the module 20 . Genes with the highest eigengene-based connectivity were selected as hub genes, and presented together with their expression levels (Fig. 6). The largest changes in differential expression were observed during the first hours of the experiment. The transcriptional response in the first hour after the temperature increase displayed the largest log2 fold change value, a large reversal was observed for all modules after 4 h. Despite this reversal in differential expression, about half of the hub genes stay up or downregulated to a lower extent throughout the experiment. Hub genes are representative for their associated hub and therefore these hubs and their associated functions, as annotated through GO-term and KEGG pathway enrichment, hypothetically display a comparable expression pattern (Supplementary material 4). In general, the results from the WGCNA correspond to enrichment analysis results. Several interesting hub genes were identified which play a role in the response to an increase in temperature.

Discussion
Cell physiology changes severely after exposure to supra-optimal temperature. In an industrial photobioreactor, temperature can raise to levels that significantly affect the growth and productivity of microalgae. Microalgae are continuously exposed to changing environmental conditions throughout the day and between days. We aimed to characterize the effect of supra-optimal temperature on the physiology of the green microalga Picochlorum sp. (BPE23). We subjected Picochlorum sp. (BPE23) to a supra-optimal temperature of 42 °C for 120 h, whereas 38 °C is the optimal temperature for growth 14 . Cell growth, cell volume, pigment composition, fatty acid composition and mRNA expression were measured throughout the experiment. While www.nature.com/scientificreports/ such long exposure to high temperature does not commonly occur in nature or in photobioreactors, this study generated knowledge on how microalgae immediately cope and eventually acclimate to supra-optimal temperatures. Two different sequential response phases were observed after the temperature shift from 30 to 42 °C. In the first phase (0-8 h), the cell cycle was arrested and an increase in cell volume was observed. The cell cycle resumed after 8 h as cells started to divide and returned to their original cell size. During the second phase (8-120 h), Picochlorum sp. (BPE23) acclimated towards a new homeostatic phase. The average cell volume gradually increased between 24 and 120 h. This increase in cell size was unexpected as increased temperature commonly causes a decreased cell volume in other microalgal species to counteract the imbalance between catabolic and anabolic processes 1 . The growth rate and cell composition stabilized after 24-72 h. The changes in mRNA transcription were very severe between 0 and 4 h, while between 4 and 120 h differential expression levels rapidly became smaller.
Interestingly, the hub gene in the red module, minichromosome maintenance (MCM2/3/5) family protein, was downregulated 5 times log2 fold during the first hours after temperature increase. The MCM complex is critical for cell division and essential for DNA replication as it is a target of various checkpoint pathways required for S-phase entry 21 . This confirms the hypothesis that the cell cycle was arrested to protect the DNA from temperature-induced damage during the replication cycle. In literature, the cell cycle of Chlamydomonas reinhardtii was found to be inhibited after a heat shock treatment 4,12 . Full recovery of the cell cycle and recovery of the cell physiology in C. reinhardtii was only observed after the temperature was decreased.
Heat shock proteins were upregulated directly after the temperature increase. A heat shock response was observed in the first hours, and to a lesser extent throughout the experiment. HSP20 and HSP70 were found among the strongest upregulated genes in the dataset. HSP70 was the main cause for enrichment in the endocytosis, protein processing and the endoplasmic reticulum pathways. Moreover, genes encoding for HSP40, HSP60, and HSP90, and HSFs showed upregulated gene expression. HSFs are transcriptional activators that regulate expression of heat shock proteins. The heat shock proteins protect cells from adverse effects of thermal stress through chaperoning and refolding of polypeptides and by stabilizing protein complexes, cellular membranes and key cellular processes 5,6,22 . Their regulatory role provides thermotolerance and the ability to gradually acclimate to a new environmental temperature by mediating the part of the cellular stress response that deals with protein homeostasis 23 . Next to the high upregulation of heat shock proteins, most of the other genes in the endocytosis pathway were also upregulated (~ 0.5-1 times log2 fold). In addition to regulation by heat shock proteins, the 5.3 times log2 fold upregulation of the ubiquitin-like superfamily protein-encoding genes is the major cause for the enrichment of the autophagy pathway and is involved in numerous stress-related functions such as the cell cycle, DNA repair, transcription, autophagy, and post-translational protein modification 24 . Proteins can still become damaged despite chaperoning and stabilization. Degradation of damaged proteins and proteins that are no longer required for cell functioning is essential to maintain cellular homeostasis 2 . The 20S proteasome alpha subunit F2 (PAF2) encoding gene was upregulated 7.5 times log2 fold and is involved in the degradation of proteins with partially unfolded regions 25 .
DNA repair in microalgae is still poorly understood 2,6 . However, heat shock proteins are known to initiate DNA repair after stressful events. HSP70 and HSP27 in particular are associated to this activity 26 . DNA replication, base excision repair, Nucleotide excision repair, and mismatch repair were enriched in the downregulated www.nature.com/scientificreports/ genes at 1 h, whereas DNA replication was enriched in the downregulated genes at 24 h and 120 h. The DNA associated network interference module (red) shows a similar pattern of downregulation, followed by almost complete reversal towards a log2 fold change value of ~ 0. Hypothesizing from the mRNA expression data, DNA was not damaged after exposure to supra-optimal temperatures, nor by formation of ROS.

Supra-optimal temperature downregulates photosynthesis but increases pigment concentrations.. The quantum yield (F v /F m ) increased during the first hours after the temperature increase, indicat-
ing an increased efficiency of photosystem II at 42 °C. After 8 h, the quantum yield decreased until the end of the experiment. Moreover, between 8 and 120 h, gene expression of nuclear genes encoding subunits of both photosystem I and photosystem II was enriched in the downregulated genes. mRNA expression for genes associated to both photosystems was downregulated despite the fact that photosystems are not considered a bottleneck at survivable but supra-optimal culture temperatures. In addition, several genes in the Porphyrin and chlorophyll metabolism show up to fourfold downregulation, of which the most severely downregulates genes are: Coproporphyrinogen III, Uroporphyrinogen decarboxylase, and NAD(P)-binding Rossman-fold superfamily protein. Genes enriched in the turquoise hub, associated with chloroplasts, plastids, and the thylakoid membrane, showed downregulation throughout the experiment. Research on Dunaliella bardawil reported that photosynthesis was severely downregulated and that chlorophyll content decreased dramatically after exposure to a heat treatment of 42 °C for 2 h, after which temperature was returned to the optimal temperature 13 . In Picochlorum sp. (BPE23), the mRNA transcript levels indicate overall decreased photosynthetic activity. However, an unexpected increase in chlorophyll-a and chlorophyll-b was observed nonetheless. Downregulation of photosynthesis is done to reduce the formation of ROS 13 . When metabolism becomes unbalanced due to temperature stress the excessive energy flux from photosynthesis results in the formation of ROS. Unfortunately, too few genes in the carotenoid biosynthesis KEGG pathway gained a functional annotation. Therefore, little insight on the transcriptional response of the carotenoid biosynthesis pathway was gained. However, data on pigment concentration in the biomass show that the concentration of the carotenoids: zeaxanthin, violaxanthin, lutein, β-carotene and canthaxanthin increased as a result of exposure to supra-optimal temperature. An increase in carotenoid content in response to temperature increase was also found in the literature for D. bardawil and Haematococcus pluvialis 13,27 . Increased levels of carotenoids are often induced when microalgae are exposed to abiotic stress conditions such as salinity, light, and temperature for quenching of ROS 10,28 .
The metabolism and protein synthesis were downregulated to conserve energy. As expected, connected to downregulation of genes associated to photosynthesis, the carbon fixation pathway and central energy metabolism were downregulated as determined through KEGG pathway enrichment. All analyses point towards downregulation of the metabolism of Picochlorum sp. in response to temperature stress. Lower photosynthetic activity ultimately results in depletion of the cell's energy reserves. Suppression of ribosome biogenesis reduces the energy demand of the cell through reduction of protein synthesis to conserve energy 25 . Ribosomal activity was significantly downregulated throughout the experiment. In a study with a thermosensitive Arabidopsis Thaliana strain it was hypothesized that the ribosomal activity was downregulated in response to stressful temperature to prevent errors in translation and protein folding 29 . Production of misfolded proteins would not only lead to wasted energy, but could also lead to formation of toxic compounds in the cell that cause further damage 8 . Therefore, reduced ribosomal activity indirectly relieves the workload of chaperones. Other protein processing related processes were affected throughout the experiment. The rate of protein synthesis was adapted to the reduced growth rate that was caused by the increased temperature. At the same time, the conserved energy was available for damage repair caused by oxidative stress and acclimation to the new temperature 6 . Downregulation of protein production and photosynthesis after temperature stress was also observed in Picochlorum costavermella 29 . We observed that downregulation of these vital cellular processes led to a severe decrease in growth rate.

Picochlorum sp. (BPE23) accumulated fatty acids under heat stress. The concentration of neu-
tral fatty acids is initially low (3.0 mg g −1 ) as cells are grown in nitrogen replete growth medium 30 . However, 24 h after temperature increase, a fatty acid concentration of 11.5 mg g −1 was measured. This significant 383% increase shows the funnelling of excess energy into de novo synthesis of lipid bodies as energy storage. In a study on Chlamydomonas reinhardtii and Chlorella vulgaris a screening was done for neutral lipid accumulation at elevated temperature, after which about 25% of the mutant strains showed similar elevated lipid accumulation 31 . Apparently, induction of neutral lipids through increased temperature is strain specific. Next to neutral lipids, also the polar membrane fatty acid concentration increased, from 79.0 to 88.5 mg g −1 . Especially C16:0 increased significantly. A study on Chlamydomonas reinhardtii showed that translocation of unsaturated fatty acids from cell membranes to neutral lipids happened after a temperature increase, to maintain optimal membrane fluidity 4 . Especially plastid membranes are heat sensitive, and quick modification of these membranes is necessary to keep photosynthesis running 32 . Almost all hubs found through the WGCNA analysis show gene ontology annotation to the plastid membranes, indicating that these membranes are also severely affected in Picochlorum sp. (BPE23).
Genes in the fatty acid biosynthesis pathway exhibited downregulation as an overall trend from 0 to 1 h, although the pathway itself was not significantly enriched due to the presence of few severely upregulated genes, up to 2.6 times log2 fold (Supplementary material 6). Pathview pathway analysis indicated increased mRNA expression of genes encoding for Malonyl-CoA synthase, which catalyses the reaction from malonate into malonyl-CoA. In addition expression of mRNA of acyl carrier protein and long chain acyl-CoA synthase were upregulated. However, drawing accurate conclusions is challenging as the fatty acid biosynthesis pathway is only partly annotated in the used reference genome of Picochlorum sp. SENEW3 (assembly ASM87641v1) 33 . www.nature.com/scientificreports/ Another possible reason for the fatty acid accumulation could be high stability of enzymes involved in fatty acid biosynthesis which resulted in the continuation of fatty acid biosynthesis, whereas other metabolic activity and cell growth decreased. Although the total increase in fatty acid content was just 18 mg g −1 at 24 h and 24 mg g −1 at 72 h, this increase corresponds to a percentual gain in fatty acid content of 22% and 38%, respectively. The ability to induce an increased fatty acid concentration by increasing temperature for only 24 h opens up the possibility to boost the lipid content of microalgal biomass before harvest.

Conclusions
In this study, the mechanisms underlying temperature stress and acclimation were determined through assessment of cell physiology and transcriptome analysis. A cell cycle arrest and a transcriptomic heat shock response were observed directly after an increase in temperature to 42 °C, 4° above the optimal growth temperature. During the first 8 h, various regulatory and chaperoning mechanisms were differentially expressed to protect the cells from heat-induced cell damage. Between 8 and 120 h after temperature increase, an acclimation process towards a new homeostatic phase started. Photosynthesis, carbon fixation, ribosomal activity, and central metabolism were downregulated, hypothetically to reduce oxidative cell damage. At the same time, pigment concentration increased significantly and the concentration of fatty acids increased by 22% over between 0 and 24 h. The combination of these effects led to a decrease in growth rate throughout the experiment. This study contributes knowledge on the effect of supra-optimal temperature on the physiology of the industrially relevant green microalgae Picochlorum sp. (BPE23).

Materials and methods:
Cell cultivation. Growth media and inoculum preparation. Picochlorum sp. (BPE23), isolated from a saltwater body of Bonaire was pre-cultivated in shake flasks in an orbital shaker incubator (Multitron, Infors HT) with a 12/12 h day/night cycle and a light intensity of 100 μmolph m −2 s −114 . The temperature was 30 °C during night and 40 °C during day. Furthermore, the relative humidity of the air in the incubator was set to 60% and enriched with 2% CO 2 . Cells were cultured in artificial seawater enriched with nutrients and trace elements. Elements were provided at the following concentrations (in g L −1 ): NaCl, 24.   www.nature.com/scientificreports/ Biomass harvest and lyophilizing. Biomass samples for compositional analysis were taken at the same moment as when offline measurements were performed. Microalgae cells were pelleted by centrifugation at 4000g for 5 min and washed with 0.5 M ammonium formate. The centrifugation/washing cycle was repeated twice more after which the cell pellet was frozen at − 20 °C. Samples were then lyophilised for 24 h and stored at − 20 °C until further processing.
Fatty acid analysis. Fatty acids within the triacylglycerol (TAG) and polar lipids (PL) fraction were quantified through GC-FID analysis according to 35  Transcript assembly and annotation. Paired-end reads were mapped to the genome of Picochlorum sp. SENEW3 (assembly ASM87641v1) using HISAT2 v2.2.1 with the -very-sensitive pre-set 33,37 . Transcript were assembled and predicted using StringTie v2.1.4, which was guided by the structural annotation of Picochlorum sp. SENEW3. StringTie's prepDE3 python script was used to extract gene counts and predict genes de novo. Functional annotation was initiated by a BLASTP search of the translated Picochlorum sp. (BPE23) coding sequences against the protein sequences of Arabidopsis thaliana with an E-value threshold of 1E −10 . Unannotated genes were filtered and orthology inference was conducted using OrthoFinder v2.5.2 against the protein sequences of the microalgae Auxenochlorella protothecoides, Chlorella variabilis, Chlamydomonas reinhardtii, and Helicosporidium sp. Ortholog gene identifiers were then matched to their gene description. Functional annotation was concluded by matching unannotated genes to their inferred domains, as derived from Pico-PLAZA.
Differential expression analysis and GO, and KEGG enrichment analysis. Pairwise differential expression (DE) analysis was performed using the DESeq2 v1.30.0. R package. Sample-level quality control consisted of pairwise correlation clustering, hierarchical clustering, and Principal Component Analysis (PCA). Fold change values were generated on a log2 scale (LFC). Two designs for data display were implemented; first, where a control condition after 0 h was used for each sample to compare DE between stressed and non-stressed growth. Second, where the previous sampling moment was used as a control condition to compare DE over time. Genes with a false discovery rate (FDR) adjusted p value ≤ 0.05 and an LFC > 1 were considered as significantly differentially expressed.
Arabidopsis thaliana gene identifiers that matched to Picochlorum sp. (BPE23) genes were linked to their corresponding DE-analysis results and used for GO and KEGG pathway enrichment analysis 38 . Enrichment analyses were conducted by use of clusterProfiler v3.18.1. and org.At.tain.db v3.12.0. packages. GO-terms and KEGG pathways with an FDR-adjusted p value ≤ 0.05 and a positive or negative enrichment score were considered as significantly enriched and visualized with the ggplots2 package. www.nature.com/scientificreports/ Network interference analysis. Weighted gene co-expression network analysis (WGCNA) was conducted by applying the WGCNA R package v1.69 20 . The correlation network was inferred from a correlation matrix of normalized counts. The optimal value of power was determined through scale-free topology analysis. The network was restricted to genes with informative connectivity, referring to a connectivity higher than the median connectivity of the entire network. Modules were then constructed with average linkage hierarchical clustering using distances in the topological overlap construction. Networks were constructed in a hybrid adaptive tree with a deepSplit of 1, a power of 12, a minimum cluster size of 30, a cut height of 0.8, and no PAM-like stage filtering. Subsequently, the gene with the highest eigengene-based connectivity was identified for each module and considered the module's hub gene. Furthermore, modules were annotated with GO-terms and KEGG pathways to infer functional properties.  27-30 (2000).